First discovery of parasite eggs in a vertebrate coprolite of the Late Triassic in Thailand

A paleoparasitological investigation of a vertebrate coprolite from the Huai Hin Lat Formation (Upper Triassic) was carried out. Five morphotypes of potential parasite eggs or sporocysts were identified in the coprolite by microscopic analysis using thin section technique. The rounded or oval shape and thick shell of one of the five morphotypes suggests that it belongs to nematode of the order Ascaridida. Systematic assignment of other morphotypes cannot be done in detail but suggests that the host was parasitized by different species of parasites. This is the first record of parasites in terrestrial vertebrate hosts from the Late Triassic in Asia and it provides new information on parasite-host interactions during the Mesozoic era.


Materials and methods
The studied coprolite was photographed, measured, and classified based on its shape. To search for internal structures and fossil inclusion, the coprolite was hardened by embedding in epoxy resin, and then cut with a diamond saw in longitudinal and transversal sections using a standard thin section method. The coprolite slices were glued to glass slides, and optimal thickness for transmission microscopy was obtained using a grinder with a graded series [6, 44]. All microscopic structures and fossil remains were photographed with a light microscope Nikon ECLIPSE E200, and multiple images taken with different focal distances were combined using a focus stacking technique.

Results
The coprolite has an elongated cylindrical shape, curved on one side, with a rounded end ( Fig  2), and is approximately 74 mm in length and 21 mm in diameter. The surface is hard, smooth and grey in colour. Microscopic observations of all slides showed a dark, high-density clay-like material and absence of soft tissue, e.g., folded or spiral traces. Five different morphotypes of organic structures were visible in the coprolite slices. Morphotype I: ellipsoid in shape with a round to oval sections (Fig 3) and 40 to 60 μm wide and 50 to 70 μm long. They exhibit a hardly discernible and relatively thin shell (1 to 2μm) and

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First discovery of parasite eggs in a vertebrate coprolite of the Late Triassic in Thailand show internal structures that could correspond to dividing cytoplasmic or nuclear material ( Fig 3A). The most elongated one ( Fig 3D) shows an apical opening (micropyle or operculum).
Morphotype II: spherical in shape (Fig 4) with a diameter of around 80 μm and a thick (3-4 μm) and irregular shell that is interrupted and could present a pore; the surface shows slightly developed wrinkles.
Morphotype III: irregular ellipsoid shape (Fig 5) with a minimum diameter of 80 μm and a maximal diameter of 120 μm) with a very thick shell (10 μm) and it shows a segmented or multicellular body within the shell.
Morphotype IV: spherical rounded shape (Fig 6) with a clearly defined shell of 3 to 5 μm, with a reticulated and anastomosed surface ornamentation. This morphotype is 80 to 140 μm in diameter. Within that structure, no clear cellular or nuclear material can be observed and there is no evidence of pore or operculum.
Morphotype V ellipsoid shape (Fig 7) with a very thick shell (7 to 9 μm). It is 60 μm long and 43 μm wide. In this morphotype, a second translucent layer is observable within the shell and is irregular in thickness (2 to 7 μm). There is no evidence of operculum, and the ornamentation of external surface is not properly appreciated.

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First discovery of parasite eggs in a vertebrate coprolite of the Late Triassic in Thailand

Discussion and systematic assessment
The sedimentological conditions of the site where the coprolite was found allowed the preservation of parasites and other organic structures in the coprolite. Microscopic observation of thin sections revealed the highly well-preserved internal structures of the organic structures. This approach had yielded similar results in a study of tapeworm eggs in a shark coprolite [6].
The five observed morphotypes are potentially eggs of parasite. Morphotypes II and IV present some ornamentation on external surfaces. No clearly organized bodies could be defined within these structures. Ornamented surfaces could initially correspond to the sporoderm of fern or moss spores that might have been ingested but not digested by the animal. However, our sections are not showing any fern or moss spore diagnostic features (structures are not triangular or kidney shaped, there is no indication of aperture or sporogenesis scars). The wrinkled surface could correspond to the diagnostic surface of nematode eggs. Using chemical methods may have provided information the external characteristic ornamentation, as in the studies of fossilized Ascaridida eggs using chemical methods [3,5,8,45], but they cannot provide important information on internal structures.
Morphotypes I, III and V apparently do not present trace of ornamentation and are likely not plant spores but rather parasite elimination form through the feces of their hosts. A large

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First discovery of parasite eggs in a vertebrate coprolite of the Late Triassic in Thailand number of parasite groups carry out these elimination steps (protists and helminths). Morphotype I is relatively small and could potentially correspond to unicellular cyst (i.e., Coccidia). Protist cysts have been found in terrestrial coprolites dating back to the early Cretaceous period [45]. Oocysts of coccidians, such as Eimeria, overlap in size range with this morphotype [46,47]. Morphotype III shows a well-developed shell and organized bodies within the shell. It could be a nematode egg with a developed embryo, but confirmation requires other diagnostic characters.
The ellipsoid shape and thick wall of morphotype V are diagnostic traits of eggs of Ascaridida nematodes [48]. Parasites of this order are commonly found in terrestrial vertebrates such as fishes, amphibians, reptiles, birds, mammal-like reptiles, and mammals [3,5,8,49,50]. Fossil eggs of nematodes have been ascribed to the genus Ascarites. The earliest fossil record of Ascarididae eggs, ascribed to Ascarites rufferi is Triassic and comes from a cynodont coprolite from Rio Grande do Sul State in Brazil [5]. Other Mesozoic findings are Ascarites gerus and Ascarites priscus [45] from Early Cretaceous archosaur coprolites (iguanodontian dinosaur) in Belgium, and Ascaridida eggs discovered in Crocodyliformes coprolites from the Early Cretaceous in Brazil [3]. Morphotype V differs from Ascarites priscus by its smoother shell and slightly larger size and by its more homogeneous vitelline layers, it differs also from Ascarites gerus and A rufferi by its smooth and thicker shell. Other individual eggs must be found and studied using both thin sections and chemical techniques to be able to create a new taxon.
The cylindrical and curved shape of the coprolite and the absence of prey remains are typical of the Crurotarsi, especially crocodile-like animals [3,20,51,52]. Crocodiles are not known in Huai Hin Lat but abundant vertebrate assemblages have been discovered in several outcrops of the Huai Hin Lat Formation including actinopterygian fishes [25,34,35], lungfish [36,37],

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First discovery of parasite eggs in a vertebrate coprolite of the Late Triassic in Thailand temnospondyls [25,27,38,39], turtles [40,41], and phytosaurs [42]. The studied coprolite was therefore likely produced by a crocodile-like reptile, possibly a phytosaur, a reptile that evolved convergently with crocodilians, and whose tooth and bone remains have been found in the same formation [25,42,43].
The discovery of at least six parasites with at least five different morphotypes in a single coprolite suggests that multi-parasite infection was common had already diversified by the late Triassic. The presence of the Ascaridida eggs and the evidence for multi-infection found in the coprolite can presumably be explained by the predatory habits of the host, which would have been parasitized by feeding on parasitized fishes, amphibians, or other reptiles [3,4,18].

Conclusion
Parasites of several species, including Ascaridida eggs were found in a coprolite probably produced by a crocodile-like reptile and possibly a phytosaur. This is therefore the first discovery of Ascaridida eggs and evidence of multi-infection in a host assignable to the Crurotarsi from the Late Triassic of Asia.